Preparation of nucleotide-free elongation factor Tu and its stabilization by the antibiotic kirromycin

A rapid, highly reproducible procedure for the preparation of nucleotide-free elongation factor Tu (EF-Tu) is described. A microscale gel filtration is performed in a two-step elution, which takes less than 30 min and allows the preparation of nanomole amounts of the factor. The nucleotide-free EF-Tu is unstable, as measured by its ability to bind GDP. However, it can be stabilized either by the presence of a residual contamination of GDP of at least 1%, in the absence of Mg²⁺, or by kirromycin. In the presence of the latter component, the nucleotide-free EF-Tu is stable over a long period of time, similarly to the EF-Tu· GDP complex. Both GDP and kirromycin promote the reactivation of partially inactivated nucleotide-free EF-Tu, as measured by GDP binding and GTPase activity.